High-throughput, massively parallel nucleotide sequence analysis has made in-depth studies of the human microbiome feasible. Thus far, most microbiome studies have focused on bacteria, although some include fungi. Viruses are particularly understudied, in part due to the challenges of assessing their presence in clinical samples. Viruses as a group have highly variable genomes, with no gene shared among all viruses that can be surveyed by an amplicon-based sequencing strategy. Therefore, studies of viruses based on nucleotide sequencing require a metagenomic approach. Metagenomic shotgun sequencing (MSS) is a relatively unbiased, culture-independent method in which nucleic acid extracted from a sample is sequenced. Sequence reads are classified based on similarity to reference genomes. This approach allows comprehensive study of the viral component of the microbiome (the virome) and has led to the discovery of novel viruses and the characterization of viruses present in healthy and sick people. When adequate numbers of sequence reads are generated, viruses can be characterized with regard to taxonomy and the presence of genes associated with virulence and resistance to antiviral drugs.
A limitation of MSS as employed to date for virus detection is that the amount and proportion of viral nucleic acid in samples from humans may be very low, and in these cases, few viral sequences are generated. Additionally, it has been shown that viruses known to be present based on molecular assays are undetectable using MSS. These difficulties may reflect the small genome size of some viruses and/or low levels of virus in the sample. This can be a particular problem for studies of the virome of healthy, asymptomatic individuals, in whom virus levels may be low. Thus, there is a need for a method of enriching viral sequences in a metagenomic sample prior to sequencing.